A crystalline form and b crystalline form of pyrazine-2(1h)-ketone compound and preparation method thereof

ABSTRACT

Provided are crystalline forms of pyrazine-2(1H)-ketone compound and a preparation method thereof; specifically disclosed are a method for preparing the compound of formula (II) and crystalline

The present application claims the following priorities:

CN201910731663.3, Aug. 8, 2019;

CN201911059460.0, Nov. 1, 2019.

TECHNICAL FIELD

The present disclosure relates to a crystal form of a pyrazin-2(1H)-one compound and a preparation method therefor, specifically relates to a preparation method of a compound of formula (II) and a crystal form thereof.

BACKGROUND

Fibroblast growth factor receptor (FGFR) is a receptor for fibroblast growth factor (FGF) signal transduction; the family of FGFR consists of four members (FGFR1, FGFR2, FGFR3, FGFR4) and FGFR is a glycoprotein consisting of an extracellular immunoglobulin (Ig)-like domain, a hydrophobic transmembrane region and an intracellular portion including a tyrosine kinase region. Fibroblast growth factor (FGF) plays an important role in many physiological regulatory processes such as cell proliferation, cell differentiation, cell migration and angiogenesis through these receptors (FGFR). There is much evidence linking FGF signaling pathway abnormalities (high expression, gene amplification, gene mutations, chromosomal reorganization, etc.) directly to many pathological processes such as tumor cell proliferation, migration, invasion, and angiogenesis. Therefore, FGFR has emerged as an important class of therapeutic targets that has attracted extensive research and development interest.

CONTENT OF THE PRESENT INVENTION

The present disclosure provides a crystal form A of a compound of formula (II), and the X-ray powder diffraction pattern thereof comprises characteristic diffraction peaks at the following 2θ angles: 4.99±0.20°, 9.54±0.20°, and 10.45±0.20°.

In some embodiments of the present disclosure, the X-ray powder diffraction pattern of the crystal form A of the compound of formula (II) comprises characteristic diffraction peaks at the following 2θ angles: 4.99±0.20°, 5.85±0.20°, 9.54±0.20°, 10.45±0.20°, 11.45±0.20°, 13.19±0.20°, 15.93±0.20°, and 19.48±0.20°.

In some embodiments of the present disclosure, the X-ray powder diffraction pattern of the crystal form A of the compound of formula (II) comprises characteristic diffraction peaks at the following 2θ angles: 4.99°, 5.85°, 9.54°, 9.94°, 10.45°, 11.45°, 13.19°, 13.92°, 14.51°, 15.11°, 15.93°, 16.86°, 17.94°, 19.48°, 19.90°, 20.80°, 21.60°, 21.97° 23.28°, 23.66°, 25.52°, 26.19°, 27.47°, 28.66°, 29.03° and 29.42°.

In some embodiments of the present disclosure, the XRPD pattern of the crystal form A of the compound of formula (II) is as shown in FIG. 1.

In some embodiments of the present disclosure, the analytical data of the XRPD pattern of the crystal form A of the compound of formula (II) are as shown in Table 1.

TABLE 1 Analytical data of the XRPD pattern of the crystal form A of the compound of formula (II) Angle Relative of 2θ d-Spacing intensity No. (°) (Å) (%)  1 4.988 17.7033 100.0  2 5.853 15.0861 12.6  3 9.539 9.2639 85.5  4 9.936 8.8944 14.7  5 10.446 8.4618 50.1  6 11.447 7.7236 19.5  7 13.194 6.7046 18.0  8 13.916 6.3586 6.1  9 14.513 6.0981 8.5 10 15.106 5.8603 6.0 11 15.933 5.5577 10.0 12 16.858 5.2549 6.3 13 17.944 4.9393 3.9 14 19.485 4.5518 19.2 15 19.905 4.4569 11.4 16 20.804 4.2662 13.5 17 21.596 4.1116 7.0 18 21.968 4.0427 10.5 19 23.281 3.8176 5.5 20 23.660 3.7573 4.9 21 25.522 3.4873 15.3 22 26.190 3.3998 8.1 23 27.468 3.2444 4.5 24 28.658 3.1124 10.3 25 29.032 3.0732 9.7 26 29.425 3.0330 5.1

In some embodiments of the present disclosure, the differential scanning calorimetry curve of the crystal form A of the compound of formula (II) has an endothermic peak with an onset at 201.7° C.±2.0° C., and an endothermic peak with an onset at 254.8° C.±2.0° C.

In some embodiments of the present disclosure, the DSC pattern of the crystal form A of the compound of formula (II) is as shown in FIG. 2.

In some embodiments of the present disclosure, the thermogravimetric analysis curve of the crystal form A of the compound of formula (II) shows a weight loss of 0.5876% occurred at 63.0° C.±3.0° C., a weight loss of 2.4156% occurred at 127.4° C.±3.0° C., a weight loss of 8.8666% occurred at 209.4° C.±3.0° C., and a weight loss of 11.8416% occurred at 263.2° C.±3.0° C.

In some embodiments of the present disclosure, the TGA pattern of the crystal form A of the compound of formula (II) is as shown in FIG. 3.

The present disclosure provides a crystal form B of the compound of formula (II), and the X-ray powder diffraction pattern thereof comprises characteristic diffraction peaks at the following 2θ angles: 10.57±0.20°, 14.59±0.20°, and 16.05±0.20°.

In some embodiments of the present disclosure, the X-ray powder diffraction pattern of the crystal form B of the compound of formula (II) comprises characteristic diffraction peaks at the following 2θ angles: 4.60±0.20°, 10.57±0.20°, 13.82±0.20°, 14.59±0.20°, 16.05±0.20°, 19.52±0.20°, 21.45±0.20°, and 27.16±0.20°.

In some embodiments of the present disclosure, the X-ray powder diffraction pattern of the crystal form B of the compound of formula (II) comprises characteristic diffraction peaks at the following 2θ angles: 4.60°, 5.87°, 9.06°, 9.54°, 10.57°, 11.42°, 13.21°, 13.82°, 14.59°, 16.05°, 17.23°, 18.16°, 19.52°, 20.12°, 20.22°, 21.45°, 22.00°, 22.95°, 23.61°, 24.06°, 24.61°, 25.85°, 26.29°, 27.16°, 28.44°, 28.68°, 30.94°, 32.07°, 32.40°, 33.01°, 33.94° 34.82°, 35.64°, 36.95° and 38.88°.

In some embodiments of the present disclosure, the XRPD pattern of the crystal form B of the compound of formula (II) is as shown in FIG. 4.

In some embodiments of the present disclosure, the analytical data of the XRPD pattern of the crystal form B of the compound of formula (II) are as shown in Table 2.

TABLE 2 Analytical data of the XRPD pattern of the crystal form B of the compound of formula (II) Angle Relative of 2θ d-Spacing intensity No. (°) (Å) (%)  1 4.596 19.2099 9.6  2 5.874 15.0334 5.7  3 9.056 9.7565 4.3  4 9.538 9.2649 3.6  5 10.567 8.3653 100.0  6 11.416 7.7449 7.4  7 13.210 6.6967 6.6  8 13.822 6.4015 11.3  9 14.590 6.0663 36.7 10 16.050 5.5175 14.5 11 17.232 5.1416 1.3 12 18.161 4.8806 5.0 13 19.516 4.5449 8.4 14 20.120 4.4097 2.0 15 20.217 4.3887 1.8 16 21.454 4.1384 10.1 17 21.997 4.0375 2.1 18 22.950 3.8719 6.6 19 23.611 3.7650 3.1 20 24.056 3.6963 3.0 21 24.609 3.6145 2.6 22 25.852 3.4434 5.8 23 26.286 3.3875 7.2 24 27.156 3.2810 12.2 25 28.439 3.1359 4.8 26 28.676 3.1104 4.6 27 30.941 2.8878 1.7 28 32.068 2.7888 2.7 29 32.400 2.7610 1.8 30 33.010 2.7113 1.2 31 33.935 2.6395 2.5 32 34.816 2.5747 1.2 33 35.638 2.5171 1.3 34 36.954 2.4305 1.1 35 38.876 2.3147 1.0 / / / /

In some embodiments of the present disclosure, the differential scanning calorimetry curve of the crystal form B of the compound of formula (II) has an endothermic peak with an onset at 224.4° C.±2.0° C., and an endothermic peak with an onset at 257.4° C.±2.0° C.

In some embodiments of the present disclosure, the DSC pattern of the crystal form B of the compound of formula (II) is as shown in FIG. 5.

In some embodiments of the present disclosure, the thermogravimetric analysis curve of the crystal form B of the compound of formula (II) shows a weight loss of 0.1558% occurred at 90.9° C.±3.0° C., a weight loss of 6.6758% occurred at 206.5° C.±3.0° C., and a weight loss of 9.4648% occurred at 255.9° C.±3.0° C.

In some embodiments of the present disclosure, the TGA pattern of the crystal form B of the compound of formula (II) is as shown in FIG. 6.

The present disclosure also provides a use of the crystal form A and the crystal form B in the manufacture of a medicament for treating diseases related to FGFR.

Technical Effect

The crystal form A and the crystal form B of the compound of formula (II) are stable and easy to form drugs. According to the experimental embodiments of the trifluoroacetate salt of the compound of formula (I), it can be seen that the crystal form A and the crystal form B of the compound of formula (II) exhibit better inhibitory activity against wild-type FGFR, and the selectivity of FGFR2 and 3 against FGFR1 and 4 is higher. The pharmacokinetic index of the crystal form A and the crystal form B of the compound of formula (II) in mice is good.

The trifluoroacetate salt of the compound of formula (I) exhibits better inhibitory activity against wild-type FGFR, and the selectivity of FGFR2 and 3 against FGFR1 and 4 is higher. The pharmacokinetic index of the trifluoroacetate salt of the compound of formula (I) in mice is good.

Definition and Description

Unless otherwise indicated, the following terms and phrases used in this document are intended to have the following meanings. A specific term or phrase should not be considered indefinite or unclear in the absence of a particular definition, but should be understood in the ordinary sense. When a trade name appears herein, it is intended to refer to its corresponding commodity or active ingredient thereof.

The compounds of the present disclosure can be prepared by various synthetic methods known to those skilled in the art, including the embodiments described below, the embodiments formed by combining the embodiments described below with other chemical synthesis methods, and equivalent alternatives well-known to those skilled in the art, preferred embodiments include, but are not limited to, the embodiments of the present disclosure.

The chemical reactions of the embodiments of the present disclosure are carried out in a suitable solvent, and the solvent should be suitable for the chemical change of the present disclosure, and the reagents and materials required. In order to obtain the compounds of the present disclosure, it is sometimes necessary for those skilled in the art to modify or select the synthetic steps or reaction schemes based on the existing embodiments.

The present disclosure will be specifically described below by way of embodiments, but the scope of the present disclosure is not limited thereto.

All solvents used in the present disclosure are commercially available and can be directly used without further purification.

The following abbreviations are used in the present disclosure: eq refers to equivalent, equivalents; PE refers to petroleum ether; DMSO refers to dimethyl sulfoxide; MeOH refers to methanol; and TFA refers to trifluoroacetic acid.

The solvents used in the present disclosure are commercially available and the commercially available compounds use their vendor directory names. When the mixed solvents are added to the reaction solution, each solvent can be mixed first and then added to the reaction solution; or each solvent can be added to the reaction solution in turn and mixed in the reaction system.

Compounds are named according to conventional naming principles in the field or by ChemDraw® software, and the commercially available compounds use their vendor directory names.

X-Ray Powder Diffractometer (XRPD) Method in the Present Disclosure

About 10 to 20 mg of the sample was subjected to XRPD detection.

Detailed XRPD parameters are as follows:

X-ray tube: Cu, k2, (λ=1.54056{acute over (Å)})

X-ray tube voltage: 40 kV, X-ray tube current: 40 mA

Divergence slit: 0.60 mm

Detector slit: 10.50 mm

Anti-scattering slit: 7.10 mm

Scanning range: 4-40 deg

Step size: 0.02 deg

Step time: 0.12 second

Rotation speed of sample tray: 15 rpm

Differential Scanning Calorimeter (DSC) Method in the Present Disclosure

Samples (0.5 to 1 mg) were placed in a DSC aluminum crucible for testing, and the method was: 25° C. to 300 or 350° C., 10° C./min.

Thermal Gravimetric Analyzer (TGA) Method in the Present Disclosure

Samples (2 to 5 mg) were placed in a TGA platinum crucible for testing, under the condition of 25 mL/min N₂ at a heating rate of 10° C./min, the sample was heated from room temperature to 300° C. or until a weight loss of 20%.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is the XRPD pattern of the crystal form A of the compound of formula (II) measured by Cu-Kα radiation.

FIG. 2 is the DSC pattern of the crystal form A of the compound of formula (II).

FIG. 3 is the TGA pattern of the crystal form A of the compound of formula (II).

FIG. 4 is the XRPD pattern of the crystal form B of the compound of formula (II) measured by Cu-Kα radiation.

FIG. 5 is the DSC pattern of the crystal form B of the compound of formula (II).

FIG. 6 is the TGA pattern of the crystal form B of the compound of formula (II).

DETAILED DESCRIPTION OF THE EMBODIMENT

The present disclosure is described in detail below by way of embodiments, but no adverse limitation of the disclosure is implied. The present disclosure has been described in detail herein, wherein specific embodiments thereof are also disclosed, and it will be apparent to those skilled in the art that various variations and improvements can be made to specific embodiments of the present invention without departing from the spirit and scope of the disclosure.

Embodiment 1: Preparation of the Compound of Formula (I) and the Trifluoroacetate Salt Thereof

Step 1: Synthesis of Compound BB-1-3

Compound BB-1-2 (2.0 g, 11.49 mmol, 1 eq) and compound BB-1-1 (2.6 g, 11.49 mmol, 1 eq) were dissolved in water (6.0 mL) and dioxane (25.0 mL), followed by the addition of [1,1′-bis(diphenylphosphino)ferrocene] palladium dichloride (841 mg, 1.15 mmol, 0.1 eq) and potassium carbonate (4.8 g, 34.48 mmol, 3 eq), and the mixture was heated to 100° C. and reacted for 16 hours under the protection of nitrogen. The obtained reaction mixture was filtered under reduced pressure and evaporated to dryness by rotary evaporation, and the crude product was purified by column chromatography (petroleum ether:ethyl acetate=1:0 to 0:1) to obtain compound BB-1-3.

MS (ESI) m/z: 190.0[M+H]⁺.

Step 2: Synthesis of Compound BB-1

Compound BB-1-3 (0.5 g, 2.64 mmol, 1 eq) and pyridine (209 mg, 2.64 mmol, 213.28 μL, 1 eq) were added to chloroform (20.0 mL), and the mixture was cooled to 0° C., and then bromine (422 mg, 2.64 mmol, 136.22 μL, 1 eq) was added thereto. The reaction was carried out at room temperature of 28° C. for 18 hours. The reaction was quenched with sodium thiosulfate (1.0 mL), then filtered under reduced pressure, and the filtrate was concentrated, and the crude product was purified by flash silica gel column chromatography (petroleum ether:ethyl acetate=1:0 to 1:1). Compound BB-1 was obtained. MS (ESI) m/z: 267.9[M+H]⁺.

¹H NMR (400 MHz, CD₃OD) δ: 8.12 (s, 1H), 7.90 (s, 1H), 3.86 (s, 3H), 2.43 (s, 3H).

Step 3: Synthesis of Compound 2

Under the protection of nitrogen, compound BB-2-1 (2.0 g, 18.77 mmol, 2.17 mL, 1 eq, HCl) was dissolved in chlorobenzene (15.0 mL), and then compound BB-2-2 (8.3 g, 65.69 mmol, 5.8 mL, 3.5 eq) was added dropwise at 25° C.; the mixture was slowly heated to 90° C. and stirred for 16 hours. Water (30.0 mL) and ethyl acetate (30.0 mL) were added to the reaction system, and the mixture was stood and then the layers were separated, and the aqueous phase was extracted three times with ethyl acetate (20.0 mL, 20.0 mL, 20.0 mL). The organic phases were combined, washed once with saturated sodium chloride solution (30.0 mL), and finally dried with anhydrous sodium sulfate, filtered and concentrated under reduced pressure. The crude product was separated and purified by column chromatography (petroleum ether:ethyl acetate=1:0 to 2:1) to obtain compound 2. MS (ESI) m/z: 178.7[M+1]⁺.

¹H NMR (400 MHz, CDCl₃) δ: 7.26 (s, 1H), 3.61 (s, 3H).

Step 4: Synthesis of Compound 4

Compound 2 (0.2 g, 1.12 mmol, 1 eq) and compound 3 (213 mg, 1.17 mmol, 1.05 eq) were dissolved in a mixed solution of dioxane (1.5 mL) and water (1.5 mL) in a microwave tube under the protection of nitrogen, and tetrakis(triphenylphosphine)palladium (65 mg, 55.86 μmol, 0.05 eq) and sodium carbonate (130 mg, 1.23 mmol, 1.1 eq) were added, and the mixture was stirred at 120° C. for 30 minutes under microwave irradiation. The reaction mixture was concentrated directly. The crude product was separated by column chromatography (petroleum ether:ethyl acetate=1:0 to 0:1) (TLC detection petroleum ether:ethyl acetate=1:1) to obtain compound 4. MS (ESI) m/z: 281.0[M+1]⁺.

¹H NMR (400 MHz, CDCl₃) δ: 7.64 (d, 2H), 7.28 (s, 1H), 6.59 (t, 1H), 3.86 (s, 6H), 3.61 (s, 3H).

Step 5: Synthesis of Compound 0027-1

Compound 4 (250 mg, 890.61 μmol, 1 eq) was dissolved in a mixed solution of acetonitrile (20.0 mL) and dichloromethane (5.0 mL) under the protection of nitrogen; a solution of sulfonyl chloride (84 mg, 623.43 μmol, 62.33 μL, 0.7 eq) in acetonitrile (2.5 mL) was slowly added dropwise at 0° C., and the mixture was stirred at 0° C. for 10 minutes. The reaction was quenched by adding methanol (5.0 mL) to the reaction mixture and then the reaction mixture was concentrated to dryness under reduced pressure. The crude product was separated by column chromatography (petroleum ether:ethyl acetate=1:0 to 1:1) (TLC detection petroleum ether:ethyl acetate=1:1) to obtain compound 0027-1. MS (ESI) m/z: 314.9[M+H]⁺.

Step 6: Synthesis of the Compound of Formula (I)

In a three-necked flask, compound 0027-1 (59 mg, 186.49 μmol, 1 eq), bis(pinacolato)diboron (52 mg, 205.14 μmol, 1.1 eq), palladium acetate (5 mg, 20.51 μmol, 0.11 eq), 2-dicyclohexylphosphino-2,4,6-triisopropylbiphenyl (20 mg, 41.03 μmol, 0.22 eq), and potassium acetate (60 mg, 615.42 μmol, 3.3 eq) were added to a solution of dioxane (4.0 mL); the air in the reaction system was replaced with nitrogen; under nitrogen saturation, the reaction system was raised to 100° C. and stirred under reflux for 30 min, cooled to 25° C., then compound BB-1 (50 mg, 186.49 mol, 1 eq), dichloromethane complex of [1,1′-bis(diphenylphosphino)ferrocene] palladium dichloride (15 mg, 18.65 μmol, 0.1 eq), potassium carbonate (77 mg, 559.47 μmol, 3 eq), dioxane (4.0 mL) and water (2.0 mL) were added; the air in the reaction system was replaced with nitrogen; under nitrogen saturation, the temperature was raised to 100° C. and the mixture was stirred under reflux for 8 hours. The reaction mixture was concentrated directly. The obtained crude product was separated and purified by high performance liquid chromatography (chromatographic column: Boston Green ODS 150×30 mm 5 m; mobile phase: [Water (0.1% TFA)-ACN]; B %: 30% to 60%, 8 min), and the trifluoroacetate salt of the compound of formula (I) was obtained. MS (ESI) m/z: 468.2[M+H]⁺. ¹H NMR (400 MHz, CD₃OD) δ: 8.79 (s, 1H), 8.09 (m, 2H), 6.76 (m, 2H), 3.93 (s, 3H), 3.89 (s, 3H), 3.84 (s, 3H), 3.80 (s, 3H), 2.54 (s, 3H). The salt was dissolved in dichloromethane, and the mixture was washed with saturated sodium carbonate; the organic phase was dried over anhydrous sodium sulfate, filtered, and the filtrate was evaporated to dryness by rotary evaporation to obtain the compound of formula (I). ¹H NMR (400 MHz, CDCl₃) δ: 8.51 (s, 1H), 8.15 (s, 1H), 6.71 (d, J=2.8 Hz, 1H), 6.63 (d, J=2.8 Hz, 1H), 6.43 (brs, 2H), 3.94 (s, 3H), 3.92 (s, 3H), 3.84 (s, 3H), 3.79 (s, 3H), 2.55 (s, 3H).

Embodiment 2: Preparation of the Compound of Formula (II)

The compound of formula (I) (44.4 g, 94.89 mmol, 1 eq) was dissolved in tetrahydrofuran (450 mL), followed by the dropwise addition of a solution of hydrogen chloride (4 M, 94.89 mL, 4 eq) in ethyl acetate, and the mixture was stirred at 25° C. for 3 hours. The reaction mixture was filtered to obtain a yellow solid, which was dried by an oil pump. The compound of formula (II) was obtained. ¹H NMR (400 MHz, DMSO-d₆) δ: 8.71 (s, 1H), 8.18 (s, 2H), 6.82 (d, J=2.8 Hz, 1H), 6.75 (d, J=2.8 Hz, 1H) 3.91 (s, 3H), 3.81 (s, 3H), 3.80 (s, 3H), 3.71 (s, 3H), 2.44 (s, 3H).

Embodiment 3: Preparation of the Crystal Form a of the Compound of Formula (II)

400 mg of the compound of formula (II) was weighed and placed into a 40 mL glass flask, and 20 mL of ethanol was added thereto, and then the mixture was stirred to form a suspension. The sample was placed on a magnetic stirrer (50° C.) and was stirred for 60 hours (protected from light). The sample was centrifuged quickly, and the residual solid was put into a vacuum drying oven, and vacuum-dried at 45° C. overnight to remove the residual solvent to obtain the crystal form A.

Embodiment 4: Preparation of the Crystal Form B of the Compound of Formula (II)

400 mg of the compound of formula (II) was weighed and placed into a 40 mL glass flask, and 20 mL of ethyl acetate was added thereto, and then the mixture was stirred to form a suspension. The sample was placed on a magnetic stirrer (50° C.) and was stirred for 60 hours (protected from light). The sample was centrifuged quickly, and the residual solid was put into a vacuum drying oven, and vacuum-dried at 45° C. overnight to remove the residual solvent to obtain the crystal form B of the compound of formula (II).

Experimental Embodiment 1: Evaluation of Wild-Type Kinase Inhibitory Activity In Vitro

The IC₅₀ values were measured by ³³P isotope-labeled kinase activity test (Reaction Biology Corp) to evaluate the inhibitory ability of the tested compounds on human FGFR1 and FGFR4.

Buffer conditions: 20 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (Hepes) (pH 7.5), 10 mM MgCl₂, 1 mM ethylene glycol-bis-(2-aminoethyl ether) tetraacetic acid (EGTA), 0.02% polyoxyethylene lauryl ether (Brij35), 0.02 mg/mL bovine serum albumin (BSA), 0.1 mM sodium vanadate (Na₃VO₄), 2 mM dithiothreitol (DTT), 1% DMSO.

Experimental steps: At room temperature, the tested compounds were dissolved in DMSO to prepare a 10 mM solution for later use. The substrate was dissolved in the freshly prepared buffer, and the tested kinase was added thereto and the mixture was mixed well. The DMSO solution dissolved with the tested compound was added to the above-mixed reaction mixture using acoustic technique (Echo 550). The concentrations of compounds in the reaction mixture were 10 μM, 3.33 μM, 1.11 μM, 0.370 μM, 0.123 μM, 41.2 nM, 13.7 nM, 4.57 nM, 1.52 nM, 0.508 nM, or 10 μM, 2.50 μM, 0.62 μM, 0.156 μM, 39.1 nM, 9.8 nM, 2.4 nM, 0.61 nM, 0.15 nM, 0.038 nM. After 15 minutes of incubation, ³³P-ATP (activity 0.01 μCi/μL, corresponding concentrations are listed in Table 3) was added to start the reaction. The supplier number, lot number and concentration information in the reaction mixture of FGFR1, FGFR4 and their substrates are listed in Table 3. After incubating the kinase reaction for 120 minutes at room temperature, the reaction mixture was deposited on P81 ion exchange filter paper (Whatman #3698-915). After repeatedly washing the filter paper with 0.75% phosphoric acid solution, the radioactivity of the phosphorylated substrate remaining on the filter paper was measured. The kinase activity data were expressed by the comparison of the kinase activity of the test compound with the kinase activity of the blank group (containing DMSO only). The IC₅₀ value was obtained by curve fitting with Prism4 software (GraphPad). The experimental results are shown in Table 4.

TABLE 3 Related information of kinase, substrate and ATP in test in vitro. Kinase Supplier Cat# Lot # ATP concentration (μM) FGFR1 Invitrogen PV3146 28427Q 5 FGFR2 Invitrogen PV3368 31517I 5 FGFR3 Invitrogen PV3145 28459R 30 FGFR4 Invitrogen P3054 26967J 2.5 Concentration of substrate in Substrate Supplier Cat# Lot # reaction mixture (μM) pEY (mg/mL) + Mn Sigma P7244-250 MG 062K5104V 0.2 pEY (mg/mL) + Mn Sigma P7244-250 MG 062K5104V 0.2 pEY (mg/mL) + Mn Sigma P7244-250 MG 062K5104V 0.2 pEY (mg/mL) + Mn Sigma P7244-250 MG 062K5104V 0.2

TABLE 4 Results of in vitro screening tests for compounds of the present disclosure IC₅₀ (nM) Selectivity Compound FGFR1 FGFR2 FGFR3 FGFR4 FGFR½ FGFR⅓ The trifluoroacetate salt 2.12 0.53 0.69 106 4.02 3.06 of the compound of formula (I) Conclusion: The trifluoroacetate salt of the compound of formula (I) exhibits better inhibitory activity against wild-type FGFR, and the selectivity of FGFR2 and 3 against FGFR1 and 4 was higher.

Experimental Embodiment 2: Pharmacokinetic Evaluation of Compounds

Experimental Purposes: To Test the Pharmacokinetics of the Compound in Mice

Experimental Materials:

CD-1 mice (male), vehicle (0.5% (w/v) methylcellulose 0.5% (v/v) Tween 80 aqueous solution), trifluoroacetate salt of compound 0027.

1. Preparation of Formulation for Administration:

The vehicle was 0.5% (w/v) methylcellulose 0.5% (v/v) Tween 80 aqueous solution and was prepared according to the following procedure:

a. About 50% volume of pure water was added to a suitable container and heated to about 60° C. to 70° C.

b. When the water temperature reached the specified value range, the heater was turned off. The required amount of methylcellulose was slowly added to the container and the mixture was stirred continuously.

c. The mixture was stirred continuously at 4° C. until the mixture was visually clarified.

d. The required volume of Tween 80 was added to the above solution. The mixture was stirred continuously until Tween 80 was visually dispersed and clarified.

e. The above solution was fixed to a final volume with an appropriate amount of pure water.

f. The mixture was stirred continuously until a homogeneous solution was formed.

Preparation of Formulation for Intragastric Administration:

a. An appropriate amount of the test product was weighed into a glass flask;

b. 70% volume of vehicle (0.5% (w/v) methylcellulose 0.5% (v/v) Tween 80 aqueous solution) were added thereto;

c. the formulation was stirred until visually homogeneous, with water bath sonication when required;

d. the remaining volume of 0.5% methylcellulose and 0.5% Tween 80 were supplemented and the mixture was stirred uniformly.

2. Administration

Animals in groups 1 and 2 were given 5 mg/mL and 30 mg/mL compound by single intragastric administration, respectively, with a dose volume of 10 mL/kg.

The animals were weighed before administration, and the administration volume was calculated according to the body weight.

3. Sample Collection and Processing

Whole blood samples (30 μL) were collected at specified times (0.25, 0.5, 1, 2, 4, 6, 8, 24 hours) by saphenous vein collection and the actual time of collection was recorded in the test record. The acceptable error for the collection time points was ±1 minute for time points within 1 hour of administration and ±5% of the theoretical time for other time points.

All blood samples were immediately transferred to labeled commercial centrifuge tubes containing K2-EDTA. After blood samples were collected, the blood samples were centrifugated at 4° C. and 3200 rpm for 10 minutes, and the supernatant plasma was aspirated, and then quickly put into dry ice at a temperature of −20° C. or lower for LC-MS/MS analysis. And the pharmacokinetic parameters were calculated, and experimental result: See Table 5.

TABLE 5 Results of the pharmacokinetics test The trifluoroacetate salt of the compound of formula (I) Compound dosage Parameter 50 mpk 300 mpk C_(max) (nM) 14800 42100 T_(max) (hr) 1.00 7.00 T_(1/2) (hr) 2.46 ND T_(last) (hr) ND 24.0 AUC_(0-last) 85826 699413 (nM · hr) AUC_(0-inf) 95847 ND (nM · hr) MRT_(0-last) (h) 4.33 11.1 MRT_(0-inf) (h) 5.39 ND ND refers to: undetermined

Conclusion: The pharmacokinetic index of the trifluoroacetate salt of the compound of formula (I) in mice is good. 

What is claimed is:
 1. A crystal form A of a compound of formula (II), wherein the X-ray powder diffraction pattern thereof comprises characteristic diffraction peaks at the following 2θ angles: 4.99±0.20°, 9.54±0.20°, and 10.45±0.20°,


2. The crystal form A as defined in claim 1, wherein the X-ray powder diffraction pattern thereof comprises characteristic diffraction peaks at the following 2θ angles: 4.99±0.20°, 5.85±0.20°, 9.54±0.20°, 10.45±0.20°, 11.45±0.20°, 13.19±0.20°, 15.93±0.20° and 19.48±0.20°.
 3. The crystal form A as defined in claim 2, wherein the X-ray powder diffraction pattern thereof comprises characteristic diffraction peaks at the following 2θ angles: 4.99° 5.85°, 9.54° 9.94° 10.45°, 11.45°, 13.19°, 13.92°, 14.51°, 15.11°, 15.93°, 16.86°, 17.94°, 19.48°, 19.90°, 20.80°, 21.60°, 21.97°, 23.28°, 23.66°, 25.52°, 26.19°, 27.47°, 28.66°, 29.03° and 29.42°.
 4. The crystal form A as defined in claim 3, wherein the X-ray powder diffraction pattern thereof is as shown in FIG.
 1. 5. The crystal form A as defined in claim 1, wherein the differential scanning calorimetry curve thereof has an endothermic peak with an onset at 201.7° C.±2.0° C. and has an endothermic peak with an onset at 254.8° C.±2.0° C.
 6. The crystal form A as defined in claim 5, wherein the DSC pattern thereof is as shown in FIG.
 2. 7. The crystal form A as defined in claim 1, wherein the thermogravimetric analysis curve thereof shows a weight loss of 0.5876% occurred at 63.0° C.±3.0° C., a weight loss of 2.4156% occurred at 127.4° C.±3.0° C., a weight loss of 8.8666% occurred at 209.4° C.±3.0° C., and a weight loss of 11.8416% occurred at 263.2° C.±3.0° C.
 8. The crystal form A as defined in claim 7, wherein the TGA pattern thereof is as shown in FIG.
 3. 9. A crystal form B of the compound of formula (II), wherein the X-ray powder diffraction pattern thereof comprises characteristic diffraction peaks at the following 2θ angles: 10.57±0.20°, 14.59±0.20°, and 16.05±0.20°


10. The crystal form B as defined in claim 9, wherein the X-ray powder diffraction pattern thereof comprises characteristic diffraction peaks at the following 2θ angles: 4.60±0.20°, 10.57±0.20°, 13.82±0.20°, 14.59±0.20°, 16.05±0.20°, 19.52±0.20°, 21.45±0.20° and 27.16±0.20°.
 11. The crystal form B as defined in claim 10, wherein the X-ray powder diffraction pattern thereof comprises characteristic diffraction peaks at the following 2θ angles: 4.60°, 5.87°, 9.06°, 9.54°, 10.57°, 11.42°, 13.21°, 13.82°, 14.59°, 16.05°, 17.23°, 18.16°, 19.52°, 20.12°, 20.22°, 21.45°, 22.00°, 22.95°, 23.61°, 24.06°, 24.61°, 25.85°, 26.29°, 27.16°, 28.44°, 28.68°, 30.94°, 32.07°, 32.40°, 33.01°, 33.94°, 34.82°, 35.64°, 36.95° and 38.88°.
 12. The crystal form B as defined in claim 11, wherein the XRPD pattern thereof is as shown in FIG.
 4. 13. The crystal form B as defined in claim 9, wherein the differential scanning calorimetry curve thereof has an endothermic peak with an onset at 224.4° C.±2.0° C. and has an endothermic peak with an onset at 257.4° C.±2.0° C.
 14. The crystal form B as defined in claim 13, wherein the DSC pattern thereof is as shown in FIG.
 5. 15. The crystal form B as defined in claim 9, wherein the thermogravimetric analysis curve thereof shows a weight loss of 0.1558% occurred at 90.9° C.±3.0° C., a weight loss of 6.6758% occurred at 206.5° C.±3.0° C. and a weight loss of 9.4648% occurred at 255.9° C.±3.0° C.
 16. The crystal form B as defined in claim 15, wherein the TGA pattern thereof is as shown in FIG.
 6. 17. (canceled) 